Process to prepare malayamycin derivatives

ABSTRACT

A process is provided for the preparation of a compound of the general formula (I): wherein R is H or C 1-4  alkyl, which comprises treating a compound of the general formula (II): where R″ is R or R 8 CO, R is as defined above, R 8  is C 1-8  alkyl or optionally substituted phenyl and R 9  is optionally substituted C 1-8  alkyl or optionally substituted aryl, with an amine R′″NH 2  wherein R′″ is H or C 1-4  alkyl. Also provided are the trans isomers of the compound (I) where R is CH 3  (6-epi-malayamycin A) and H (6-epi-desmethylmalayamycin A), the cis and trans isomers of the compound (I) where R is C 2-4 alkyl and various intermediate compounds.

This invention relates to a process for the preparation of biocidalcompounds. More particularly it relates to a synthetic method forpreparing biocidal compounds, certain of which are novel and certain ofwhich have only previously been obtained by cultivation of Streptomycesorganisms. It also relates to novel intermediates used in the process.

Biocidal compounds of the formula (A):

where R′ is H or CH₃, and their isolation from a fermentation broth of astrain of micro-organism from the species Streptomyces malaysiensis aredescribed in International patent application No. PCT/GB2003/000063. Thecompounds of formula (A) have six asymmetric centres and may exist inthe form of one or more isomers. Particularly mentioned inPCT/GB2003/000063 are the compounds of formulae (B) and (C), which arenamed malayamycin A and desmethylmalayamycin A, respectively.

These compounds are biocidal agents, showing antiviral and anti-cancerproperties. They are, however, of particular interest as antifungalagents, especially against plant pathogenic fungi.

The present invention provides a synthetic route to malayamycin A anddesmethylmalayamycin A and to certain of their isomers and analogues.

Thus according to the present invention there is provided a process forthe preparation of a compound of the general formula (I):

wherein R is H or C₁₋₄ alkyl (especially methyl), which comprisestreating a compound of the general formula (II):

wherein R″ is R or R⁸CO, R is as defined above, R⁸ is C₁₋₈ alkyl oroptionally substituted phenyl and R⁹ is optionally substituted C₁₋₈alkyl or optionally substituted aryl, with an amine R′″NH₂ wherein R′″is H or C₁₋₄ alkyl.

In the compound of general formula (I), the RO group may be cis or transto the NH₂CONH group. The cis isomer of the compound where R is CH₃ ismalayamycin A and the cis isomer of the compound where R is H isdesmethylmalayamycin A. The trans isomer of the compound where R is CH₃(6-epi-malayamycin A) and the trans isomer of the compound where R is H(6-epi-desmethylmalayamycin A) are novel compounds and form a furtherpart of this invention as to do both the cis and trans isomers of thecompounds where R is C₂₋₄ alkyl.

Throughout this specification, the number of carbon atoms that alkylmoieties may contain is usually stated. Where unstated, alkyl moietiesmay contain from 1 to 8, suitably from 1 to 6 and typically from 1 to 4,carbon atoms. In all cases they may be in the form of straight orbranched chains. Examples are methyl, ethyl, n- and iso-propyl, n-,sec-, iso- and tert-butyl, n-pentyl and n-hexyl. Suitable optionalsubstituents of alkyl moieties include halo (e.g. chloro, bromo andfluoro), C₁₋₆ alkoxy and C₁₋₆ alkylthio.

Aryl is usually phenyl. Optional substituents of aryl and aryl moietiessuch as benzyl include C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆alkoxy, C₂₋₆ alkenyloxy, C₂₋₄ alkynyloxy, halo(C₁₋₆)alkyl,halo(C₁₋₆)alkoxy, C₁₋₆ alkylthio, halo(C₁₋₆)alkylthio, C₁₋₄alkoxy(C₁₋₆)alkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl(C₁₋₄)alkyl, phenoxy,benzyloxy, benzoyloxy, cyano, nitro, —CONR^(a)R^(b), —SO₂R^(a),—OSO₂R^(a), —COR^(a), —CR^(a)═NR^(b) or —N═CR^(a)R^(b), in which R^(a)and R^(b) are independently hydrogen, C₁₋₄ alkyl, halo(C₁₋₄)alkyl, C₁₋₄alkoxy, halo(C₁₋₄)alkoxy, C₁₋₄ alkyl-thio, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyl(C₁₋₄)alkyl, phenyl or benzyl.

R⁸ is typically tert-butyl and R⁹ is typically C₁₋₄ alkyl, e.g. methyl,substituted with halo. A particular example of R⁹ is trichloromethyl.

In one aspect the invention includes a process as defined above whereinR is H or C₁₋₄ alkyl, R″ is R or R⁸CO, R⁸ is C₁₋₆ alkyl and R⁹ is C₁₋₆alkyl substituted with halo, especially chloro.

In another aspect the invention includes a process wherein R is H ormethyl, R″ is methyl or R⁸CO, R⁸ is tert-butyl and R⁹ istrichloromethyl.

In more detail, the compounds of the invention can be made as shown inSchemes 1 to 6.

The starting materials for the synthesis are either readily availablecommercially, for example ribonolactone (i.e. D-(+)-ribonic γ-lactone),or can be made by methods known in the literature, for example2,4-dimethoxypyrimidine (Gilbert, G. E.; Johnson, T. B. J. Am. Chem.Soc. 1930, 52, 2001) and 2,4-dimethoxy-5-iodopyrimidine (Das, B.; Kundu,N. G. Synthetic Comm. 1988, 18, 855).

In Scheme 1 ribonolactone can be converted into compounds of generalformula (3), by reaction with ketals of general formula (2), where R¹and R² are C₁₋₄ alkyl, and with catalysis by an acid such p-toluenesulphonic acid, followed by a silyl chloride of general formulaR³R⁴R⁵SiCl, where R³, R⁴, and R⁵ can be independently C₁₋₄ alkyl, orphenyl, in the presence of a suitable base such as imidazole. Compoundsof general formula (5) can be formed by reaction of compounds of generalformula (3) with compounds of general formula (4) at a temperaturebetween −78° C. and −30° C., but preferably at −78° C., in a suitablesolvent such as tetrahydrofuran (THF). Compounds of general formula (4)can be generated by treatment of 5-bromo- or5-iodo-2,4-dialkoxypyrimidine, where the alkyl group R⁶ is C₁₋₄ alkyl,with n-, s- or t-butyl lithium. Compounds of general formula (5) canexist as either the beta- or alpha-anomer, or as a mixture. Compounds ofgeneral formula (7) can be formed from compounds of general formula (5)by reduction with a suitable reducing agent such as triethylsilane, inthe presence of a Lewis acid such as boron trifluoride etherate, in asuitable solvent such as dichloromethane (DCM), followed bychromatographic separation of the mixture of anomers. Alternativelycompounds of general formula (5) can be first selectively reduced tocompounds of general formula (6), by reaction with a reducing agent suchas L-selectride, in the presence of a Lewis acid such as zinc chloride,in a suitable solvent such as DCM, at a temperature starting at −78° C.and warming to room temperature. Compounds of general formula (6) canthen be reacted under Mitsunobu conditions, for example withdiethylazodicarboxylate (DEAD) and triphenyl phosphine, in a suitablesolvent such as DCM. Compounds of general formula (8) can be formed byreaction of compounds of general formula (7) with compounds of generalformula (2), with catalysis by an acid such p-toluene sulphonic acid.Compounds of general formula (9) can be formed by de-silylation ofcompounds of general formula (8), for example with a source of fluorideion such as tetrabutylammonium fluoride, in a suitable solvent such asTHF.

In Scheme 2 compounds of general formula (10) can be formed by oxidisingcompounds of general formula (9) with a suitable oxidising agent such asdimethyl-sulphoxide and oxalyl chloride in the presence oftriethylamine. Compounds of general formula (11) can be formed bytreatment of compounds of general formula (10) with a Wittig reagentsuch as methylenetriphenylphosphorane, generated by reacting amethyltriphenylphosphonium salt with a strong base such as potassiumt-butoxide. Compounds of general formula (12) can be formed bydeprotection of compounds of general formula (11) by treatment with aweak acid such as 70% acetic acid. Compounds of general formula (13) canbe formed by reacting compounds of general formula (12) with atrialkyltin oxide of general formula R¹ ₂SnO, where R¹ is as definedabove. Compounds of general formula (14) can be formed by reaction ofcompounds of general formula (13) with a source of fluoride ion such ascaesium fluoride, in the presence of an alkylating agent R⁷LG, where R⁷is a substituted benzyl group, such as 4-methoxybenzyl, and LG is aleaving group such as chlorine or bromine. Compounds of general formula(15) can be formed from compounds of general formula (14) by reactionwith an allylating agent CH₂═CHCH₂LG, such as allyl bromide, in thepresence of a base such as sodium hydride, in a suitable solvent such asdimethylformamide (DMF). Compounds of general formula (16) can be formedfrom compounds of general formula (15) in a ring-closing metathesisreaction by treatment with the Grubbs catalyst,[(cyclohexyl)₃P]₂Cl₂Ru═CHPh where Ph is phenyl, in a suitable solventsuch as DCM, at a temperature between room temperature and 40° C.Compounds of general formula (17), where Hal is a halogen atom such aschlorine, bromine or iodine, can be formed by reaction of compounds ofgeneral formula (16) with a halogen source such as N-bromosuccinimide,in the presence of water and an organic solvent such as diethyl ether.

In Scheme 3 compounds of general formula (18) can be formed from olefinsof general formula (17) by reaction with a base such as sodium hydroxidein a suitable solvent such as THF. Compounds of general formula (19) canbe formed by treatment of compounds of general formula (18) with a metalazide MN₃ where M is for example an alkali metal, such as sodium azide,in a suitable solvent such as methoxyethanol.

Compounds of general formula (22) where the RO group, in which R is C₁₋₄alkyl, is cis to the azide group, can be formed from compounds ofgeneral formula (19) in three steps. Compounds of general formula (20)can be formed from compounds of general formula (19) by oxidation with asuitable oxidising agent such as[1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzodioxol-3-(1H)-one](Dess-Martin periodinane) in a suitable solvent such as DCM. Compoundsof general formula (21) can be formed from compounds of general formula(20) by reduction with a suitable reducing agent such as sodiumborohydride, in a suitable solvent such as methanol. Compounds ofgeneral formula (22), where the RO group is cis to the azide group, canbe formed by reaction of compounds of general formula (21) with a basesuch as sodium hydride, and a compound of general formula RLG, where Ris C₁₋₄ alkyl and LG is a leaving group. An example of RLG is methyliodide.

Compounds of general formula (22) where the RO group is trans to theazide group, can be formed by reaction of compounds of general formula(19) directly with a base such as sodium hydride, and a compound ofgeneral formula RLG, such as methyl iodide. Compounds of general formula(23), where the RO group can be either cis or trans to the azide group,can be formed from compounds of general formula (22) by deprotectionwith a reagent such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).Compounds of general formula (24), where the RO group can be either cisor trans to the azide group, can be formed from alcohols of generalformula (23) by reaction with acid chlorides of general formula R⁸COCl,where R⁸ is a C₁₋₈ alkyl or optionally substituted phenyl group, in thepresence of a base such as triethylamine and a basic catalyst such as4-dimethylamino-pyridine (DMAP), in a suitable solvent such as pyridine.

In Scheme 4 compounds of general formula (25), where the RO group can beeither cis or trans to the azide group, can be formed from compounds ofgeneral formula (24) by reaction with a deprotecting reagent such astrimethylsilyl chloride and sodium iodide in a suitable solvent such asacetonitrile. Compounds of general formula (26) where the RO group canbe either cis or trans to the amino group can be formed by reaction ofcompounds of general formula (25) with a reducing agent such as atrialkylphosphine (R¹)₃P, where R¹ is as defined above, for exampletrimethylphosphine, in a suitable solvent such as a mixture of water andTHF. Compounds of general formula (27), where the RO group is cis ortrans to the R⁹CONHCONH group can be formed by reacting compounds ofgeneral formula (26) with an isocyanate R⁹CONCO, where R⁹ can be C₁₋₈alkyl group optionally substituted with a halo group such as chlorine orbromine, for example trichloroacetyl isocyanate, or an optionallysubstituted aryl group, in a suitable solvent such as DCM.

Compounds of general formula (1) can be made from compounds of generalformula (27), where the RO group is cis or trans to the NH₂CONH group,can be made by deprotection with an amine R′″NH₂, where R′″ is asdefined above, in a suitable solvent such as a mixture of methanol andwater.

Scheme 5 shows the preparation of compounds of general formula (34),which are particular examples of compounds of general formula (I), whereR is H.

Compounds of general formula (28) can be prepared from alcohols ofgeneral formula (19) or (21) by reaction with acid chlorides of generalformula R⁸COCl, in the presence of a base such as triethylamine, and abasic catalyst such DMAP. Compounds of general formula (29) where theR⁸COO group can be cis or trans to the azide group, can be formed fromcompounds of general formula (28) by deprotection with a reagent such asDDQ, in a suitable solvent such as DCM. Compounds of general formula(30), where the R⁸COO group can be cis or trans to the azide group, canbe formed from compounds of general formula (29) by reaction with acidchlorides of general formula R⁸COCl, in the presence of a base such astriethylamine, and a basic catalyst such as DMAP. Compounds of generalformula (31), where the R⁸COO group can be cis or trans to the azidegroup, can be formed from compounds of general formula (30) by reactionwith a deprotecting reagent such as trimethylsilyl chloride and sodiumiodide in a suitable solvent such as acetonitrile. Compounds of generalformula (32), where the R⁸COO group can be cis or trans to the aminegroup, can be formed by reaction of compounds of general formula (31)with a reducing agent such as a trialkylphosphine (R¹)₃P, where R¹ is asdefined above, for example trimethylphosphine, in a suitable solventsuch as a mixture of water and THF. Compounds of general formula (33),where the R⁸COO group can be cis or trans to the R⁹CONHCONH group can beformed by reacting compounds of general formula (32) with an isocyanateR⁹CONCO, where R⁹ can be C₁₋₈ alkyl group optionally substituted with ahalo group such as chlorine or bromine, for example trichloroacetylisocyanate, or an optionally substituted aryl group, in a suitablesolvent such as DCM.

Compounds of general formula (34) can be made from compounds of generalformula (33), where the R⁸COO group is cis or trans to the NH₂CONHgroup, by deprotection with an amine R′″NH₂, for example methylamine,where R′″ is as defined above, in a suitable solvent such as a mixtureof methanol and water.

Scheme 6 shows the preparation of compounds of general formula (34),which are particular examples of compounds of general formula (I), whereR is H.

Compounds of general formula (35), where R¹⁰ is a suitable protectinggroup such as a (R¹¹)₃SiCH₂CH₂OCH₂ group where R¹¹ is C₁₋₄ alkyl, can beprepared from alcohols of general formula (19) or (21) by reaction withhalides of general formula R¹⁰Hal, where Hal is chlorine or bromine, inthe presence of a base such as iso-propyldiethylamine, at between roomtemperature and 80° C., but preferably between 30° C. and 50° C., in asuitable solvent such as DCM. Compounds of general formula (36) wherethe R¹⁰O can be cis or trans to the azide group, can be formed fromcompounds of general formula (35) by deprotection with a reagent such asDDQ, in a suitable solvent such as DCM. Compounds of general formula(37), where the R¹⁰O group can be cis or trans to the azide group, canbe formed from compounds of general formula (36) by reaction with acidchlorides of general formula R⁸COCl, in the presence of a base such astriethylamine, and a basic catalyst such as DMAP. Compounds of generalformula (38), where the R¹⁰O group can be cis or trans to the azidegroup, can be formed from compounds of general formula (37) by reactionwith a deprotecting reagent such as trimethylsilyl chloride and sodiumiodide in a suitable solvent such as acetonitrile. Compounds of generalformula (39), where the R¹⁰O group can be cis or trans to the aminegroup, can be formed by reaction of compounds of general formula (38)with a reducing agent such as a trialkylphosphine (R¹)₃P, where R¹ is asdefined above, for example trimethylphosphine, in a suitable solventsuch as a mixture of water and THF. Compounds of general formula (40),where the R¹⁰O group can be cis or trans to the R⁹CONHCONH group can beformed by reacting compounds of general formula (39) with an isocyanateR⁹CONCO, where R⁹ can be C₁₋₈ alkyl group optionally substituted with ahalo group such as chlorine or bromine, for example trichloroacetylisocyanate, or an optionally substituted aryl group, in a suitablesolvent such as DCM. Compounds of general formula (41) can be made fromcompounds of general formula (40), by reaction with a source of fluorideion such as boron trifluoride etherate, between 0° C. and roomtemperature, in a suitable solvent such as DCM.

Compounds of general formula (34) can be made from compounds of generalformula (41), where the OH group is cis or trans to the NH₂CONH group,by deprotection with an amine R′″NH₂, for example methylamine, where R′″is as defined above, in a suitable solvent such as a mixture of methanoland water

In a further aspect the invention includes the intermediate compounds(16) to (33) and (35) to (41) as defined above.

Compounds that may be made by the process of the invention areillustrated in Table 1. The compounds in Table 1 have the generalformula (I) in which R and the orientation of RO to the NH₂CONH— groupare as shown in the table. “Me” represents “methyl”. TABLE 1 (I)

Compound Orientation of No. R RO to NH₂CONH— Structure 1 Me cis

2 Me trans

3 H cis

4 H trans

The compounds Nos. 2 and 4 of Table 1 are novel and form a part of thepresent invention. The invention is further illustrated by the followingExamples, in which, in the structural formulae, Me is methyl, t-Bu istertiary butyl, Ph is phenyl, PMB is p-methoxybenzyl, DMF isN,N-dimethylformamide, THF is tetrahydrofuran, DMSO is dimethylsulphoxide, DCM is dichloromethane, DDQ is2,3-dichloro-5,6-dicycano-1,4-benzoquinone and DMAP is4-dimethylaminopyridine.

EXAMPLE 1

This Example illustrates the preparation of malayamycin A (Compound No.1 of Table 1).

Step 1

The preparation of:

D-(+)-ribonolactone (4.35 g, 29.3 mmol), 2,2-dimethoxypropane (18.1 ml,146 mmol) and pyridinium p-toluene sulfonate (195 mg, 0.79 mmol) weremixed together. The light yellow mixture was heated at 60° C. for 4hours, and concentrated. The yellow oil was taken up in ethyl acetate,washed with saturated sodium bicarbonate and with brine. The organiclayer was dried with magnesium sulphate and concentrated to give a whitesolid, which was dissolved in THF and a solution of 0.1 M hydrochloricacid (10 ml) was added. The colourless mixture was stirred for 10minutes at room temperature, concentrated to half volume, and dilutedwith ethyl acetate. The combined organic phases were washed withsaturated sodium bicarbonate and brine, dried over magnesium sulphateand evaporated to give the desired product as a white solid, which wasrecrystallized from 1:1 hexane:ethyl acetate, (3.5 g, 72%).

[α]_(D) ²⁰=+70.5 (c 1, CHCl₃) ¹H NMR (CDCl₃, 300 MHz) δ ppm: δ 1.40 (s,3H, CH₃), 1.50 (s, 3H, CH₃), 2.60 (t, 1H, OH), 3.72 (dd, 1H, H-5), 4.71(dd, 1H, H-5), 4.71 (t, 1H, H-3), 4.81 (d, 1H, H-4), 4.90 (d, 1H, H-2).¹³C NMR (CDCl₃, 300 MHz) δ ppm: 25.10, 26.09, 62.18, 76.12, 78.30,84.20, 113.98, 175.16.

Step 2

The preparation of:

The product of step 1 (3.25 g, 17 mmol), imidazole (2.25 g, 37.4 mmol)and DMF (anhydrous, 25 ml) were mixed and then t-butyldiphenylsilylchloride (4.6 g, 17 mmol) was added dropwise. The colourless mixture wasstirred at room temperature for 24 hours, and then poured into coldwater. The aqueous layer was extracted with ether, the combined organicphase was dried with magnesium sulphate, evaporated to give the desiredproduct as a white solid, which was recrystallized from hexane (7.02 g,97%), m.p. 86-88° C.

[α]_(D) ²⁰=+10.7 (c 1, CHCl₃) ¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.06 (9H,s, tBu), 1.41 (s, 3H, CH₃), 1.49 (s, 3H, CH₃), 3.77 (dd, 1H, J=1.3,J=11.4, H-5), 3.93 (dd, 1H, J=2.2, J=11.51, H-5), 4.59 (s, 1H,), 4.75(d, 1H, J=5.6), 4.91 (d, 1H, J=6.2), 7.4-7.6 (6H, Ph), 7.6-7.65 (4H,Ph). ¹³C NMR (CDCl₃, 400 MHz) δ ppm: 14.53, 23.06, 26.01, 27.17, 63.97,76.25, 78.86, 82.73, 113.55, 128.42, 128.44, 130.60, 130.61, 131.95,132.75, 135.85, 136.02, 174.51.

Step 3

The preparation of:

2,4-Dimethoxy-5-iodopyrimidine (1.53 g, 5.80 mmol) was dissolved in dryTHF (55 ml) under an argon atmosphere, and tert-butyl lithium (6.73 ml,11.44 mmol, 1.7 M in hexane) was added dropwise over five minutes at−78° C. and the reaction mixture was stirred for 30 minutes. The productof step 2 (2.25 g, 5.28 mmol) dissolved in dry THF (57 ml) was slowlyadded to the lithium reagent over 10 minutes. The mixture was stirred at−78° C. for four hours, and then at −20° C. for one hour. Brine wasadded, the organic phase was separated, extracted with diethyl ether,and the combined organic phases dried over magnesium sulphate. Theorganic fraction was evaporated to afford an oil which was purified byflash chromatography over silica eluting with 8:2 hexane:ethyl acetateto give the (1.78 g, 3.16 mmol, 60%) as a white solid, which was shownto be a 1:8 mixture of the alpha:beta anomers, with the beta-anomerbeing the desired product.Beta anomer:

M.p 51° C., [α]_(D) ²⁰=−45.3 (c 1, CHCl₃) ¹H NMR (CDCl₃, 400 MHz) δ ppm:1.08 (s, 9H, tBu), 1.17 (s, 3H, CH₃), 1.24 (s, 3H, CH₃), 3.98 (s, 3H,OMe), 4.02 (s, 3H, OMe), 4.22 (s, 1H, OH-1′), 4.35 (m, 1H, H-4′), 4.84(m, 2H, H-3′, H-2′), 7.24-7.5 (m, 6H, Ph), 7.51-7.7 (m, 4H, Ph), 8.47(s, 1H, H-6).

Step 4

The preparation of:

The product of step 3, (1 g, 1.77 mmol) was dissolved in anhydrousdichloromethane (100 ml) under an argon atmosphere. The colourlesssolution was brought to −78° C. and zinc chloride (5.31 ml, 5.31 mmol, 1M in diethyl ether) was added. The colourless mixture was stirred at−78° C. for 30 minutes and L-selectride (17.7 ml, 17.7 mmol, 1 M in THF)was added dropwise very slowly over 10 minutes at −78° C. The colourlessmixture was stirred overnight (−78° C. to room temperature slowly), andafter 18 hours, water (5 ml) was added to the yellow mixture, which wasstirred until it came colourless. After addition of 95% ethanol (15 ml),a solution of sodium hydroxide (9 ml, 6 M) was added and the mixture wascooled to 0° C. before 30% hydrogen peroxide (15 ml) was added dropwise.The mixture was stirred for 10 minutes at 0° C., then at roomtemperature for 5 minutes. The organic phase was separated and theaqueous phase was saturated with solid potassium carbonate (1 g). Theaqueous layer was extracted with ethyl acetate and the combined organicphase was dried over magnesium sulphate. The organic fraction wasevaporated to give a colourless oil (1.7 g) which was purified by flashchromatography on silica eluting with 2:1 hexane:ethyl acetate to givethe desired product as a white foamy solid (0.875 g, 1.55 mmol, 88%); asa 1:19 mixture of alpha:beta anomers.Beta anomer:

m.p.=39-41° C., [α]_(D)=−24.9 (c=0.47, CHCl₃) ¹H NMR (CDCl₃, 400 MHz) δppm: 1.09 (s, 9H, tBu), 1.30 (s, 3H, CH₃), 1.47 (s, 3H, CH₃), 2.85 (d,J=3.1, 1H, OH), 3.16 (d, J=5.1, 1H, OH), 3.83 (dd, J=3.74, J=13.9, 1H,H-5′), 3.90 (dd, J=3.80, J=13.5, 1H, H-5′), 3.96 (s, 6H, 2×OMe), 4.27(m, 2H), 4.35 (d, J=4.1, 1 H), 5.29 (s, 1 H, H-1′), 7.4 (m, 6H, Ph), 7.7(m, 4 H, Ph), 8.38 (s, 1 H, H-6)

Step 5

The preparation of:

Triphenylphosphine (0.556 g, 2.12 mmol) was added to the product of step4 (0.80 g, 1.42 mmol) in THF (anhydrous, 105 ml). The colourless mixturewas cooled to 0° C. and diethyl azodicarboxylate¹ (0.35 ml, 2.12 mmol)was added dropwise. The yellow-orange mixture was stirred overnight at4° C., then at room temperature for one hour, until no more startingmaterial was observed by TLC. The mixture was concentrated and theorange oil (1.4 g) was purified by flash chromatography eluting with 4:1hexane:ethyl acetate to give the desired product as a pure colourlessoil (0.635 mg, 1.16 mmol, 82%).

[α]_(D)=+10.5 (c=0.55 CHCl₃) ¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.06 (s, 9H,tBu), 1.35 (s, 3H, CH₃), 1.60 (s, 3H, CH₃), 3.83 (dd, J=4.6, J=11.1, 1H,H-5′), 3.88 (dd, J=3.95, J=11.23, 1H, H-5′), 3.94 (s, 3H, OMe), 3.99 (s,3H, OMe), 4.13 (m, 1H), 4.65 (dd, 1H, J=4.2, 6.4), 4.72 (dd, 1H, J=4.6,J=6.5), 4.98 (d, 1H, J=4.1, H-1′), 7.3-7.4 (m, 6H, Ph), 7.6-7.8, (m, 4H,Ph), 8.31 (s, 1H, H-6).

Step 6

The preparation of:

In a 100 ml round bottomed flask, the product of step 5 (0.940 g, 1.709mmol) was dissolved in dry THF (60 ml). A 1M solution oftetrabutylammonium fluoride in THF (1.96 ml, 1.96 mmol) was addeddropwise, at 0° C., then stirred for 45 minutes at 0° C. and 15 minutesat room temperature. The solution was then concentrated (40° C., 10mmHg) to give an oil that was purified by flash chromatography on silicaeluting with 5:5 hexane:ethyl acetate to give the title compoundquantitatively as a colorless oil, as a 1:19 mixture of alpha:betaanomers.

[α]_(D) ²⁰=−14.6° (c 0.57, CHCl₃) I.R. (Neat) ν cm⁻¹: 3397, 2990, 2938,1606, 1573, 1473, 1400, 1213, 1075, 756. ¹H NMR (CDCl₃, 400 MHz) δ ppm:1.34 (s, 3H, CH₃), 1.59 (s, 3H, CH₃), 2.35 (dd, 1H, J=3.1, J=8.6, OH),3.75 (m, 1H, H-5′), 3.87 (m, 1H, H-5′), 3.98 (s, 3H, OMe), 4.02 (s, 3H,OMe), 4.13 (m, 1H, H-4′), 4.7-4.9 (m, 3H, H-1′, H-2′, H-3′), 8.20 (s,1H, H-6). ¹³C NMR (CDCl₃, 400 MHz) δ ppm: 25.37, 27.45, 54.07, 54.85,62.46, 81.37, 81.96, 84.10, 84.29, 111.99, 114.49, 157.88, 165.33,168.69.

Step 7

The preparation of:

Oxalyl chloride (186 μL, 2.132 mmol) was added to dry dichloromethane (5ml) and the solution cooled to −78° C. under argon, and DMSO (186 μL,2.132 mmol) added dropwise. After 5 minutes, the product of step 6(0.582 g, 1.867 mmol) was added in dry dichloromethane (2 ml). After onehour at −78° C., triethylamine (1.30 ml, 95 mmol) was added dropwise andthe solution was allowed to stay at room temperature. Water (25 ml) wasadded, and the mixture was extracted with dichloromethane. The combinedorganic phases were washed with 1% hydrochloric acid (6 ml), water (10ml) and brine (10 ml). The organic phase was dried over sodium sulphate,filtered and concentrated to afford the desired product as a white solid(0.503 g, 1.625 mmol, 87%).

¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.37 (3H, CH₃), 1.59 (3H, CH₃), 3.90 (3H,CH₃), 3.99 (3H, CH₃), 4.48 (s, 1H), 4.81 (d, J=5.6), 5.03 (d, J=4.1),5.14 (s, 1H), 8.18 (1H, s), 9.55 (s, 1H).

Step 8

The preparation of:

An oven dried 100 ml round bottomed flask was charged with Ph₃PCH₃ ⁺Br⁻¹ (0.80 g, 2.23 mmol) and heated to 140° C. under vacuum (1 mm Hg)overnight. The flask was then put under an argon atmosphere and drydiethyl ether (22 ml) was added. At room temperature potassiumtert-butoxide (2.23 ml of 1M solution in THF, 2.23 mmol,) was added. Itwas stirred at room temperature for one hour, and at −40° C., theproduct of step 7 (1.625 mmol) in dry THF (3 ml) was added dropwise.After two hours at −40° C., the solution was stirred at 0° C. overnight.Then at 0° C., a saturated solution of ammonium chloride (20 ml) wasadded. The aqueous phase was extracted with diethyl ether (3×40 ml). Thecombined organic phase were dried over sodium sulphate, filtered andconcentrated. The crude oil was purified by flash chromatography undersilica eluting with 8:2, hexane:ethyl acetate to give the desiredproduct as a colourless oil (0.463 g, 1.218 mmol, 75%).

[α]_(D) ²⁰=+29.5° (c 1, CHCl₃). I.R. ν cm⁻¹: 2989, 1606, 1572, 1472. ¹HNMR (CDCl₃, 400 MHz) δ ppm: 1.32 (s, 3H, CH₃), 3.96 (s, 3H, OMe), 3.98(s, 3H, OMe), 4.35 (t, 1H, J=5.8), 4.50 (t, J=5.9, 1H), 4.68 (dd, 1H,J=3.6, J=6.5), 4.95 (d, 1H, J=3.2, H-1′), 5.23 (d, 1H, J=10.0, H-6′),5.39 (d, 1H, J=17.2, H-6′), 5.92 (m, 1H, H-5′), 8.22 (s, 1H, H-6). ¹³CNMR (CDCl₃, 400 MHz) δ ppm: 25.47, 27.44, 53.97, 54.73, 80.04, 84.82,85.15, 85.24, 112.77, 114.68, 117.57, 135.13, 156.88, 165.11, 168.51.HRMS GAB) Obtain 3081381 Calc 308.1372

Step 9

The preparation of:

The product from step 8 (1.14 g, 3.70 mmol) was dissolved in 70% aqueousacetic acid (130 ml) and the solution was refluxed for one hour. Aftercooling, the solution was concentrated and the oil was co-evaporatedtwice with toluene (20 ml). The product and dibutyltin oxide (1.14 g,5.6 mmol) in dry toluene (170 ml) were heated under reflux with removalof water during two hours. It was then concentrated, caesium fluoride(1.19 g, 7.8 mmol) was added and the flask was put under vacuum (2 mmHg) for two hours. Tetrabutylammonium iodide (0.233 g, 0.631 mmol) anddry DMF (50 ml) and finally p-methoxybenzyl chloride (511 μL, 4.5 mmol)were added. The mixture was stirred for 36 hours at room temperature,poured into a saturated solution of sodium bicarbonate (150 ml) andwater (150 ml). It was extracted with diethyl ether (5×200 ml). Thecombined organic phases were dried over sodium sulphate, filteredthrough celite and concentrated. The oil was purified by flash columnchromatography to give the desired compound as a white solid (0.502 g,35%, 1.29 mmol),

m.p.=74° C. [α]_(D) ²⁰=+23.8° (c 0.5, CHCl₃). I.R. (KBr) ν cm⁻¹: 2970,2870, 1602, 1574. NMR ¹H (CDCl₃, 400 MHz) δ ppm: 2.75 (b, 1H, OH), 3.80(s, 3H, OMe), 3.87 (m, 1H, H-3), 3.93 (dd, 1H, J=2.8, J=5.5, H-2′), 3.98(s, 3H, OMe), 4.00 (s,3H, OMe), 4.24 (dd, 1H, J=6.5, J=7.5, H-4′), 4.53(d, 1H, J=1.3, CH₂Ar), 4.70 (d, 1H, J=11.3, CH₂Ar), 5.05 (d, 1H, J=2.6,H-1′), 5.28 (d, 1H, J=10.0, H-6′), 5.44 (d, 1H, J=17.1, H-6′), 5.9-6.05(m, 1H, H-5′), 6.86 (d, 2H, J=8.6, Ar), 7.22 (d, 2H, J=8.6, Ar), 8.21(s, 1H, H-6). HRMS (MAB) obtained 388.1645; calcd 388.1634

Step 10

The preparation of

To a stirred mixture of dry DMF (1.6 ml) and sodium hydride (0.067 g,1.67 mmol, 60% in a mineral oil) was added dropwise a solution of theproduct of step 9 (0.334 g, 0.861 mmol) in dry DMF (4.6 ml) and allylbromide (161 μL, 1.86 mmol) at 0° C. When the addition was completed,the solution was stirred at room temperature under argon for two hours.A few drops of methanol were added at 0° C. and the solution was pouredinto water (50 ml). The aqueous phase was extracted with diethyl ether(4×50 ml), the combined organic phases were dried over sodium sulphate,filtered and concentrated. The oil was purified by flash columnchromatography to give desired product as a colourless oil (0.344 g,0.80 mmol, 93%).

[α]_(D) ²⁰=+37.7° (c 0.97, CHCl₃) I.R. ν cm⁻¹: 2956, 1603, 1571, 1514.¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.62 (dd, 1H, J=4.8, J=7.5, H-3′), 3.77(s, 3H, OMe), 3.92 (m, 3H, H-2, CH₂allyl), 3.97 (s, 6H, 2×OMe), 4.52 (t,1H, J=7.5, H-4), 4.59 (d, 1H, J=11.9, CH₂Ar), 4.65 (d, 1H, J=11.9,CH₂Ar), 5.15-5.3 (m, 5H, H-1, 3×CH ethylenic), 5.45 (d, 1H, J=17.1, CHethylenic), 5.75-6.0 (m, 2H, CH ethylenic), 6.82 (d, 2H, J=8.6, Ar),7.23 (d, 2H, J=8.6, Ar), 8.21 (s, 1H, H-6). ¹³C NMR (CDCl₃, 400 MHz) δppm: 53.81, 54.67, 55.10, 71.20 (2C), 78.09, ,79.43, 80.81, 81.28,113.48, 113.54, 117.28, 117.94, 129.31, 129.74, 134.14, 135.87, 156.34,159.14, 164.81, 167.85.

Step 11

The preparation of:

The product from step 10 (0.344 g, 0.80 mmol) was added to drydichloromethane (160 ml) and argon was bubbled for 20 minutes into thesolution. Then the Grubbs catalyst, [(cyclohexyl)₃P]₂Cl₂Ru═CHPh (0.039g, 0.047 mmol) was added, and a slight flow of argon was applied. Thesolution was refluxed for 5.5 hours. When the reaction was complete, themixture was concentrated to give a brown oil, which was purified byflash chromatography eluting with 8:2 hexane:ethyl acetate, to give thedesired product as a white crystalline solid (0.286 g, 0.71 mmol, 89%),m.p. 80° C.

[α]_(D) ²⁰=−33.18° (c 1.1, CHCl₃) I.R. ν cm⁻¹: 2956, 1603, 1602, 1575.¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.42 (dd, 1H, J=4.5, J=8.9, H-3′), 3.77(s, 3H, OMe), 3.93 (m, 1H, H-2′), 3.95 (s, 3H, OMe), 3.96 (s, 3H, OMe),4.40 (m, 2H, H-7′, H-7″), 4.54 (m, 1H, H-4′), 4.65 (d, 1H, J=11.9,CH₂Ar), 4.74 (d, 1H, J=11.9, CH₂Ar), 5.11 (m, 1H, H-1′), 5.69 (dm, 1H,J=10.3, H-5′), 6.28 (d, 1H, d=9.7, H-6′), 6.85 (d, 2H, J=8.7, Ar), 7.28(d, 2H, J=8.7, Ar), 8.18 (s, 1H, H-6) 13C NMR (CDCl₃, 400 MHz) δ ppm:53.87, 54.69, 55.11, 68.61, 71.28, 71.63, 78.78, 79.49, 81.72, 113.42,113.62, 127.14, 127.40, 129.14, 130.01, 156.11, 159.06, 164.84, 167.55.HRMS (MAB) obtained 401.1699; calcd 401.1710

Step 12

The preparation of:

The product from step 11 (0.133 g, 0.33 mmol) was dissolved in THF (7.4ml) and water (7.4 ml). N-bromosuccinimide (0.070 g, 0.39 mmol) wasadded to the reaction mixture, which was stirred vigorously for twohours in the dark. Then the mixture was poured into water (50 ml) (withone crystal of sodium thiosulfate) and extracted with diethyl ether(5×25 ml). The combined organic phase was dried over sodium sulphate andevaporated to give the crude desired bromo-alcohol product as a whitesolid, which was used for the next reaction without furtherpurification.

Step 13

The preparation of:

The crude bromo-alcohol product from step 12 was dissolved in THF (14ml) and sodium hydroxide (3.55 ml, 1M in water) was added. The solutionwas refluxed for 45 minutes, then poured into water (50 ml). Thesolution was extracted with diethyl ether (4×40 ml), the combinedorganic phase were dried over sodium sulphate and evaporated to give thecrude desired epoxide, which was used without further purification.

Step 14

The preparation of:

The epoxide product from step 13 was dissolved in methoxyethanol (30 ml)and sodium azide (0.327 g, 5.02 mmol) was added. The solution was heatedat 126° C. for 1.25 hours. After cooling, the solution was poured intobrine (50 ml) and extracted with diethyl ether (4×20 ml). The combinedorganic phase were dried over sodium sulphate and concentrated. The oilwas purified by chromatography on silica (7:3 hexane/ethyl acetate) togive the desired product as a white amorphous solid (0.063 mg, 0.132mmol, 41%), m.p. 119° C.

[α]_(D) ²⁰+93.1° (c 0.98, CHCl₃). I.R. ν cm⁻¹: 3400, 2915, 2105 (N₃),1604, 1572. ¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.40 b, 1H, OH), 3.58(dd, 1H,J=4.7, J=10.0, H-3′), 3.68 (m, 1H, H-6′), 3.73 (d, J=12.7, 1H, H-7),3.78 (s, 3H, OMe), 3.89 (m, 2H, H-2′, H-7′), 3.97 (s, 6H, 2×OMe), 4.33(m, 2H, H-5′, H-4′), 4.67 (s, 2H, CH₂Ar), 5.10 (m, 1H, H-1′), 6.86 (d,2H, J=8.7, Ar), 7.27 (d, 2H, J=8.7, Ar), 8.33 (s, 1H, H-6) ¹³C NMR(CDCl₃, 400 MHz) δ ppm: 53.87, 54.77, 55.12, 61.62, 68.26, 69.25, 71.41,73.32, 74.16, 79.80, 80.25, 113.26, 113.66, 129.24, 129.65, 155.76,159.18, 164.83, 167.35. HRMS (FAB) MH⁺° Calcd 460.1832; Found 460.1842

Step 15

The preparation of:

The product of step 14 (0.06 g, 0.129 mmoL) was dissolved in drydichloromethane (DCM) (3 ml) under an argon atmosphere, and[1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one] (theDess-Martin periodinane) (0.133 g, 0.42 mmol) was added. After 3.5hours, a saturated solution of sodium bicarbonate (2.5 ml) and sodiumbisulphite (2.5 ml) were added. The mixture was stirred for 10 minutes,DCM (25 ml) was added and the organic phase was lo separated and washedwith saturated sodium bicarbonate (2×10 ml). After drying over sodiumsulphate, the solution was evaporated to give the crude desired ketoneproduct as an oily residue.

Step 16

The preparation of:

The crude product of step 15 was dissolved in dry methanol (6 ml),treated with sodium borohydride (0.050 g, 1.32 mmol) at 0° C. and thesolution was stirred at room temperature for 10 minutes. The solutionwas concentrated under vacuum without heating. It was then dissolved inethyl acetate (20 ml) and washed with water (20 ml). The organic phasewas separated and the aqueous layer was extracted with ethyl acetate(2×20 ml). The combined organic phase were dried over sodium sulphate,and evaporated to give the crude desired hydroxy product as an oil.

Step 17

The preparation of:

To a stirred mixture of DMF (0.5 ml) and sodium hydride (0.019 g, 60%,0.49 mmol) at 0° C., a solution of the crude product from step 16,methyl iodide (38 μL, 5.9 mmol) in DMF (2 ml) was added dropwise. Themixture was then left at room temperature for 2 hours, a few drops ofmethanol were added at 0° C. and the solution was poured into cold water(20 ml). It was then extracted with ether (5×10 ml). The combinedorganic phase were dried over sodium sulphate and evaporated. The oilwas purified by flash column chromatography under silica (8:2hexane:ethyl acetate) to give the desired product as a white powder(0.056 g, 0.121 mmol, 93%, 3 steps), m.p. 89° C.

[α]_(D) ²⁰+60.56° (c 0.88, CHCl₃). I.R. ν cm⁻¹: 2913, 2105 (N₃), 1602,1572. ¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.47 (s, 3H, OMe), 3.61 (m, 2H,H-7′, H-6′.), 3.76 (m, 4H, OMe, H-3′), 3.85 (d, J=4.63, 1H, H-2′), 3.96(s, 3H, OMe), 3.97 (s, 3H, OMe), 4.01 (dd, J=10.04, J=2.91, 1H, H-4′),4.67 (m, 3H, CH₂Ar, H-5′), 5.16 (s, 1H, H-1′), 6.86 (d, 2H, J=8.6, Ar),7.26 (d, 2H, J=8.6, Ar), 8.44 (s, 1H, H-6) ¹³C NMR (CDCl₃, 400 MHz) δppm: 53.86, 54.76, 55.13, 57.13, 57.44, 59.72, 65.69, 71.17, 74.02,75.01, 76.41, 78.89, 81.54, 113.31, 113.66, 129.76, 156.02, 159.10,164.88, 167.23. HRMS (FAB) MH⁺ Calcd. 474.1988; Found 474.2002

Step 18

The preparation of:

The product of step 17 (0.043 g, 0.09 mmol) was dissolved in DCM (8 ml),then water (0.8 ml) and 2,3-dichloro-5,6-dicycano-1,4-benzoquinone (DDQ)(0.080 g, 0.35 mmol) were added. After one hour, saturated sodiumbicarbonate (30 ml) was added, the organic layer was separated and theaqueous phase was extracted with ethyl acetate (3×10 ml). The combinedorganic phases were washed with water (10 ml) and brine (10 ml). Afterdrying over sodium sulphate and concentrating, the oil was purified byflash chromatography (DCM then DCM:methanol, 95:5) to give the desiredcompound as a colourless oil (0.027 g, 0.076 mmol, 84%).

[α]_(D) ²⁰+93° (c 0.3, CHCl₃). IR (Neat) ν cm⁻¹: 3540, 2920, 2105, 1603,1574. ¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.49 (s, 3H, OMe), 3.57 (ddd, 1H,J=2.91, J=4.85, J=10.4, H-6′.), 3.65 (t, J=10.4, 1H, H-7′ax), 3.76 (dd,J=4.64, J=9.8, 1H, H-3′), 3.94 m(m, 2H, H-4′, H-7′eq), 3.98 (m, 4H,OMe), 4.02 (s, 3H, OMe), 4.10 (d, J=4.6, 1H, H-2′), 4.67 (m, 2H, CH₂Ar),5.06 (s, 1H, H-1′), 8.41 (s, 1H, H-6) ¹³C NMR (CDCl₃, 400 MHz) δ ppm:54.06, 54.79, 57.43, 59.40, 65.67, 73.05, 73.65, 74,67, 76.28, 83.53,112.91, 155.77, 164.41, 167.61. HRMS (FAB) MH⁺ Calcd. 353.1335; Found353.1341

Step 19

The preparation of:

The product from step 18 (0.033 g, 0.0926 mmol) was dissolved in drypyridine (1.1 ml). To this solution was added 4-dimethylaminopyridine(DMAP) (16 mg, 0.13 mmol), triethylamine (31 μL, 0.265 mmol) andpivaloyl chloride (80 μL, 0.649 mmol) and the solution was stirred for36 hours at room temperature. It was then concentrated, the residue wasdissolved ethyl acetate (20 ml) and the organic layer was washed withaqueous 1% hydrochloric acid (5 ml), followed by a saturated solution ofsodium bicarbonate (5 ml) and brine (5 ml). After drying over sodiumsulphate it was evaporated to give the crude desired pivaloyl compoundas an oil.

Step 20

The preparation of:

The crude product from step 19 was dissolved in dry acetonitrile (0.5ml). Dry sodium iodide (0.032 g, 0.21 mmol), and trimethylsilyl chloride(29 μL, 0.21 mmol) were added and the solution was stirred at roomtemperature for 16 hours. Several drops of a 10% sodium metabisulphitesolution were added until a colourless solution was obtained. Then asaturated solution of sodium bicarbonate (10 ml) was added, the aqueouslayer was extracted with ethyl actetate (5×10 ml). The combined organicphases were dried over sodium sulphate and evaporated to give an oilyresidue which was purified by flash chromatography to give the desiredproduct as a colourless oil (0.016 g, 42%, 0.0388 mmol).

[α]_(D) ²⁰+100.75° (c 0.4, CHCl₃). ¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.22,(s, 9H, tBu), 3.46 (s, 3H, OMe), 3.47 (ddd, 1H, J=2.9, J=4.0, J=10.7,H-6′.), 3.59 (t, J=10.7, 1H, H-7′ax), 3.75 (dd, J=2.7, J=10.0, 1H,H-4′), 3.86 (m, 2H, H-3′, H-7′eq), 4.65 (s, 1H, H-5′), 4.91 (s, 1H,H-1′), 5.31 (d, 1H, J=4.7, H-2′), 7.55 (s, 1H, H-6), 10.00 (br, 2H,2×NH). ¹³C NMR (CDCl₃, 400 MHz) δ 26.97, 38.70, 57.38, 59.07, 65.55,72.56, 72.81, 75.19, 76.35, 80.87, 111.63, 138.82, 152.17, 162.47,171.14. HRMS (FAB) MH⁺ Calcd. 410.1675; Found 410.1693

Step 21

Preparation of:

The product of step 20 (0.016 g, 0.0388 mmol) was dissolved in dry THF(3 ml) and argon was bubbled in the solution for 10 minutes. Water (3μL) and trimethylphosphine (44 μL, 1M solution in toluene, 0.044 mmol)were added. After 5 minutes at room temperature, the solution wasrefluxed for 30 minutes, then concentrated and held under vacuum for onehour to give the crude desired amine product, which was used withoutfurther purification.

Step 22

The preparation of:

The crude product of step 21 was dissolved in dry DCM (4 ml) andtrichloroacetylisocyanate (5 ml, 0.041 mmol) was added. After 30minutes, the solution was concentrated to give the crude desiredtrichloroacetyl urea as an oil, which was used without furtherpurification.

Step 23

The preparation of malayamycin A

The crude product from step 22 was dissolved in methanol (1 ml), 40%methylamine in water (2 ml) was added and the solution was stirred for52 hours. Concentration gave a solid that was purified by flashchromatography (9:1 DCM:methanol) to give pure malayamycin A as a whitesolid (0.008 g, 0.0233 mmol, 60%), m.p. 158° C. (dec).

[α]_(D) ²⁰+120° (c 0.19, MeOH) (authentic sample [α]_(D) ²⁰+126° (c0.36, MeOH)) ¹H NMR (D₂O, 400 MHz) identical to the authentic sample δppm: 3.30 (s, 3H, OMe), 3.38 (t, 1H, J=10.7, H-7ax′.), 3.51 (dd, J=10.7,J=5.1, 1H, H-3′), 3.69 (ddd, J=5.2, J=3.5, J=10.7, 1H, H-6′), 3.85 (dd,J=3.5, J=11.8, 1H, H-7eq′), 3.93 (dd, J=10.7, J=5.4, 1H, H-6′), 4.16 (d,1H, J=2.1, H-2′), 4.74 (s, 1H, H-1′), 4.82 (s, 1H, H-5′), 7.24 (s, 1H,H-6).

EXAMPLE 2

In this Example, data are provided for key intermediates in thepreparation of 6-epi-malayamycin A.

Intermediate 1

Intermediate 1 is prepared from the product of Step 14 in Example 1,using the same procedure as given in Step 17 for malayamycin A inExample 1.

M.p. 100° C. [α]_(D) ²⁰+53.5° (c 0.88, CHCl₃). I.R.(KBr) ν cm⁻¹: 2925,2109 (N₃), 1602, 1571. ¹H NMR (CDCl₃, 400 MHz) δ ppm: 3.34 (s, 1H,H-6′), 3.45 (s, 3H, OMe), 3.67 (m, 2H, H-7′, H-3′.), 3.79 (m, 3H, OMe),3.85 (d, J=4.51, 1H, H-2′), 3.96 (s, 3H, OMe), 3.98 (s, 3H, OMe), 4.08(d, J=12.9, 1H, H-7′), 4.35 (dd, 1H, J=3.09, J=10.01, H-4′), 4.48 (s,1H, H-5′), 4.63 (d, J=12.05, 1H, CH₂Ar), 4.77 (d, J=12.05; 1H, CH₂Ar),5.07 (s, 1H, H-1′), 6.86 (d, 2H, J=8.35, Ar), 7.29 (d, 2H, J=8.35, Ar),8.44 (s, 1H, H-6) ¹³C NMR (CDCl₃, 400 MHz) δ ppm: 54.36, 55.28, 55.67,57.75, 60.01, 66.43, 71.84, 74.15, 74.87, 77.75, 79.85, 81.10, 114.03,114.11, 129.63, 130.68, 156.33, 159.55, 165.38, 168.00. HRMS (FAB) M⁺Calcd. 473.1910; Found 474.1910

Intermediate 2

Intermediate 2 is prepared from Intermediate 1 using the same proceduregiven in Step 18 for malayamycin A in Example 1.

[α]_(D) ²⁰+78.9° (c 0.3, CHCl₃). IR (Neat) ν cm⁻¹: 3538, 2918, 2109,1602, 1571. ¹H NMR (CDCl₃, 400 MHz) δ ppm: 2.60 (br, 1H, OH), 3.33 (m,1H, H-6), 3.44 (s, 3H, OMe), 3.64 (dd, 1H, J=3.11, J=10.13, H-3′.), 3.72(dd, J=1.2, J=12.9, H, H-7′ax), 3.98 (m, 3H, OMe), 4.02 (s, 3H, OMe),4.04 (d, J=12.09, 1H, H-7′eq), 4.14 (d, J=4.86, 1H, H-2′), 4.28 (dd,J=3.21, J=10.03, 1H, H-4′), 4.49 (m, H-5, 1H), 5.00 (s, 1H, H-1l), 8.32(s, 1H, H-6) ¹³C NMR (CDCl₃, 400 MHz) δ ppm: 54.57, 55.30, 57.88, 58.65,66.65, 73.92, 74.07, 74.21, 77.29, 83.03, 113.60, 156.19, 165.49,168.33. HRMS (FAB) MH⁺ Calcd. 353.1335; Found 353.1334

Intermediate 3

Intermediate 3 is prepared from Intermediate 2 using the same proceduresgiven in Steps 19 and then 20 for malayamycin A in Example 1.

[α]_(D) ²⁰+143° (c 0.53, CHCl₃). ¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.23,(s, 9H, tBu), 3.29 (d, J=3.15, H-6, 1H), 3.37 (s, 3H, OMe), 3.59 (d, 1H,J=12.1, 1H, H-7′), 3.78 (dd, J=4.88, J=10.13, 1H, H-3′), 4.00 (d, 1H,J=13.2, H-7′eq), 4.15 (dd, J=3.24, J=10.12, 1H, H-4′), 4.43 (t, 1H,J=2.99, H-5), 4.82 (s, 1H, H-1′), 5.38 (d, J=4.93, H-2′), 7.44 (d,J=5.8, 1H, H-6), 11.02 (br, 2H, NH). ¹³C NMR (CDCl₃, 400 MHz) δ ppm:26.94, 38.74, 56.60, 59.15, 64.87, 72.74, 73.12, 73.86, 76.71, 79.60,111.70, 138.54, 152.19, 162.48, 177.55. HRMS (FAB) MH⁺ Calcd. 410.1675;Found 410.1678

6-Epi-malayamycin A

6-Epi-malayamycin A is prepared from Intermediate 3 using the sameprocedures given in Steps 21, 22, and then 23 for malayamycin A inExample 1.

[α]_(D) ²⁰+38.6° (c 0.3, MeOH). ¹H NMR (D₂O, 400 MHz) δ ppm: 3.37 (s,3H, OMe), 3.43 (s, 1H, H-6′.), 3.49 (dd, J=10.9, J=5.13, 1H, H-3′), 3.71(d, J=13.78, 1H, H-7′), 4.02 (m, 2H, H-7eq′+H-4′), 4.22 (d, 1H, J=2.5,H-2′), 4.52 (m, 1H, H-5′), 4.64 (s, 1H, H-1′), 7.33 (s, 1H, H-6).

EXAMPLE 3

This Example illustrates the fungicidal properties of certain of thenovel compounds of formula (I). The compounds were tested against avariety of foliar fungal diseases of plants. The technique employed wasas follows.

Plants were grown on an artificial, cellulose based growing medium. Thetest compounds were individually diluted in reverse osmosis water to afinal concentration of 100 ppm in water (that is, 1 mg of compound in afinal volume of 10 ml) immediately before use. TWEEN 20 (at a finalconcentration of 0.05% by volume) was added with the water to improveretention of the spray deposit. TWEEN is a registered trade mark.

The compounds were applied to the foliage of the test plants by sprayingthe plant to maximum droplet retention.

These tests were carried out against Stagonospora nodorum (LEPTNO),Blumeria graminis f. sp. tritici (ERYSGT), and Puccinia triticina(PUCCRT) on wheat. Two replicates, each containing 3 plants were usedfor each treatment. The plants were inoculated with either a calibratedfungal spore suspension or a “dusting” with dry spores 6 hours or oneday after chemical application.

After chemical application and inoculation, the plants were incubatedunder high humidity conditions (except those inoculated with Blumeriagraminis f. sp. tritici) and then put into an appropriate environment toallow infection to proceed until the disease was ready for assessment.The time period between chemical application and assessment varied fromsix to nine days according to the disease and environment. However, eachindividual disease was assessed after the same time period for allcompounds.

Assessments were carried out collectively on the plants in eachreplicate and averaged to give one result per replicate.

The disease level present (the percentage leaf area covered by activelysporulating disease) was assessed visually. For each treatment, theassessed values for all its replicates were meaned to provide meandisease values. Untreated control plants were assessed in the samemanner. The data were then processed (see formula below) to calculate aPRCO (percentage Disease Reduction from Control) value.

Banded Assessment Method and Calculation of PRCO Values

The mean disease values are banded in the manner shown below. If thedisease level value falls exactly mid-way between two of the points, theresult will be the lower of the two points.  0 = 0% disease present  1 =0.1-1% disease present  3 = 1.1-3% disease present  5 = 3.1-5% diseasepresent 10 = 5.1-10% disease present 20 = 10.1-20% disease present 30 =20.1-30% disease present 60 = 30.1-60% disease present 90 = 60.1-100%disease present

An example of a typical banded calculation is as follows:

-   -   Mean disease level for treatment A=25%

Therefore banded mean disease level for treatment A=30

-   -   Mean disease level on untreated controls=85%

Therefore banded mean disease level on untreated controls=90$\begin{matrix}{{PRCO} = {100 - {\frac{\left\{ {{Banded}\quad{mean}\quad{disease}\quad{level}\quad{for}\quad{treatment}\quad A} \right\}}{\left\{ {{Banded}\quad{mean}\quad{disease}\quad{level}\quad{on}\quad{untreated}\quad{controls}} \right\}} \times 100}}} \\{= {100 - \frac{\left( {30 \times 100} \right)}{90}}} \\{= 66.7}\end{matrix}$

The PRCO is then rounded to the nearest whole number; therefore, in thisparticular example, the PRCO result is 67.

It is possible for negative PRCO values to be obtained.

PRCO results are shown below. TABLE I COMPOUND ERYSGT PUCCRT LEPTNO NO.6 hour 1 day 1 day (Table 1) Protectant Protectant Protectant 2 50 10045 3 0 100 100Key to Table IERYSGT = Blumeria graminis triticiPUCCRT = Puccinia triticinaLEPTNO = Stagonospora nodorum

1. A process for the preparation of a compound of the general formula(I):

wherein R is H or C₁₋₄ alkyl, which comprises treating a compound of thegeneral formula (II):

wherein R″ is R or R⁸CO, R is as defined above, R⁸ is C₁₋₈ alkyl oroptionally substituted phenyl and R⁹ is optionally substituted C₁₋₈alkyl or optionally substituted aryl, with an amine R′″NH₂ wherein R′″is H or C₁₋₄ alkyl.
 2. A process according to claim 1 wherein aryl isphenyl.
 3. A process according to claim 1 wherein the optionalsubstituents of aryl and phenyl are selected from the group consistingof C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy, C₂₋₆ alkenyloxy,C₂₋₆ alkynyloxy, halo(C₁₋₆)alkyl, halo(C₁₋₆)alkoxy, C₁₋₆ alkylthio,halo(C₁₋₆)alkylthio, C₁₋₄ alkoxy(C₁₋₆)alkyl, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyl(C₁₋₄)alkyl, phenoxy, benzyloxy, benzoyloxy, cyano, nitro,—CONR^(a)R^(b), —SO₂R^(a), —OSO₂R^(a), —COR^(a), —CR^(a)═NR^(b) and—N═CR^(a)R^(b), in which R^(a) and R^(b) are independently hydrogen,C₁₋₄ alkyl, halo(C₁₋₄)alkyl, C₁₋₄ alkoxy, halo(C₁₋₄)alkoxy, C₁₋₄alkylthio, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl(C₁₋₄)alkyl, phenyl orbenzyl.
 4. A process according to claim 1 wherein R is H or C₁₋₄ alkyl,R″ is R or R⁸CO, R⁸ is C₁₋₆ alkyl and R⁹ is C₁₋₆ alkyl substituted withhalo, especially chloro.
 5. A process according to claim 1 wherein R isH or methyl, R″ is methyl or R⁸CO, R⁸ is tert-butyl and R⁹ istrichloromethyl.
 6. The trans isomer of the compound of formula (I)according to claim 1 wherein R is CH₃ (6-epi-malayamycin A) and thetrans isomer of the compound of formula (I) where R is H(6-epi-desmethylmalayamycin A).
 7. The cis and trans isomers of thecompound of formula (I) according to claim 1 wherein R is C₂₋₄ alkyl. 8.The intermediate compounds of the formulae (16) to (33) and (35) to(41):

wherein R and R⁶ are independently C₁₋₄ alkyl, R⁷ is substituted benzyl,R⁸ is C₁₋₈ alkyl or optionally substituted phenyl, R⁹ is C₁₋₈ alkyloptionally substituted with halo or is optionally substituted aryl, R¹⁰is a protecting group and Hal is halo.
 9. A compound according to claim8 wherein the substituents of benzyl and the optional substituents ofphenyl are selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy, C₂₋₆ alkenyloxy, C₂₋₆ alkynyloxy,halo(C₁₋₆)alkyl, halo(C₁₋₆)-alkoxy, C₁₋₆ alkylthio, halo(C₁₋₆)alkylthio,C₁₋₄ alkoxy(C₁₋₆)alkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl-(C₁₋₄)alkyl,phenoxy, benzyloxy, benzoyloxy, cyano, nitro, —CONR^(a)R^(b), —SO₂R^(a),—OSO₂R^(a), —COR^(a), —CR^(a)═NR^(b) and —N═CR^(a)R^(b), in whichR^(a)and R^(b) are independently hydrogen, C₁₋₄ alkyl, halo(C₁₋₄)alkyl,C₁₋₄alkoxy, halo(C₁₋₄)alkoxy, C₁₋₄ alkylthio, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyl-(C₁₋₄)alkyl, phenyl or benzyl.
 10. A compound according toclaim 8 wherein R and R⁶ are independently C₁₋₄ alkyl, R⁷ is substitutedbenzyl, R⁸ is C₁₋₆ alkyl, R⁹ is C₁₋₆ alkyl substituted with halo,especially chloro, R¹⁰ is (R¹¹)₃SiCH₂CH₂OCH₂ group where R¹¹ is C₁₋₄alkyl and Hal is chloro, bromo or iodo.
 11. A compound according toclaim 8 wherein R and R⁶ are both methyl, R⁷ is 4-methoxybenzyl, R⁸ istert-butyl, R⁹ is trichloromethyl, R¹⁰ is (CH₃)₃SiCH₂CH₂OCH₂ and Hal isbromo.